1. Field of the Invention
The present invention relates generally to the determination of total hemoglobin (Hb) in whole blood or in specifically prepared control and calibrator materials derived from whole blood, and, more particularly, to a method and reagent composition useful for performing such determinations which are free of toxic substances, such as cyanide ions.
2. Brief Description of the Prior Art
The determination of total hemoglobin is indicative of the oxygen-carrying capacity of whole blood. The reference, and most commonly used method, for determination of total hemoglobin is the cyanmethemoglobin method. In this method, ferrous ion (Fe (II)), of heme in hemoglobin, oxyhemoglobin and carboxyhemoglobin of the red blood cell is oxidized to the ferric state (Fe (III)) by ferricyanide to form methemoglobin. Methemoglobin is then combined with ionized cyanide to produce cyanmethemoglobin, which is measured photometrically at 540 nm. For further background, reference is made to Henry et al (Sds.) Clinical Chemistry, Harper & Row, Hagerstown, Md. (1974) at page 1131 et seq; Drabkin, J. Biol. Chem., 112:51 (1935); and Van Karopen and Ziljstra, Advances in Clinical Chemistry, 8:141-187.
The Hamill U.S. Pat. No. 3,874,852 discloses a reagent for hemoglobin determination, which includes ionic cyanide in an alkaline aqueous-solution having a pH of 9. In such reagents, ferricyanide is not present. Rather, the heme is oxidized to the ferric state by atmospheric oxygen. The ferric hemoglobin species then binds to the cyanide ions to produce a chromogen which is measured to quantify the hemoglobin.
Also, the Ledis et al U.S. Pat. No. 4,286,963 discloses a reagent for determining both lymphold and myeloid leukocytes and hemoglobin in whole blood. The reagent includes a surface-active quaternary ammonium salt, a phenyl or phenoxy alkanol, and a polyalcohol in an acidic buffer (pH 3.5-5.0) and does not contain ionic cyanide. The suggestion is made that-the lack of ionic cyanide is undesirable and results in instability of the chromogen formed by reaction with the hemoglobin. Ledis et al suggests that, if ionic cyanide is present in the reagent, the reagent should be at an alkaline pH for inclusion of buffered cyanide, so as to obtain a satisfactory hemoglobin derivative.
Oshiro et al, Clin. Biochem. 15 83 (1982), teach the use of a reagent for hemoglobin determination which comprises sodium dodecyl sulfate or, equivalently, sodium lauryl sulfate (SLS), an anionic surfactant, and Triton X-100, a nonionic surfactant, in a neutral buffer (pH 7.2). The red blood cells are lysed by the SLS. The presence of Triton X-100 prevents SLS from precipitating at temperatures below 5.degree. C. The reaction is completed within 5-10 minutes and produces a green chromogen having absorption maxima at 539 and 572 nm, the depth of color being indicative of the hemoglobin content.
Zander, Lang and Wolf, Clin. Chem. Acta, 136 (1984), also disclose a method for determining total hemoglobin, utilizing a reagent which consists of a nonionic surfactant, such as Triton X-100, dissolved in 0.1 N NaOH, a strongly alkaline medium. The reaction is completed within 1-2 minutes and a green chromogen is formed having an absorption maximum at 575 nm and a shoulder at 600 nm. Zander et al specifically state that the method does not function if nonionic surfactants are replaced by either cationic or anionic surfactants.
The high throughput of current automated hematologic systems require the use of methods with rapid turnover, e.g., completion time less than 30 seconds. In the case of hemoglobin determinations, such rapid turnover has only been achieved in the prior art by the use of cyanide-containing reagents of high pH and high cyanide levels. Hence, these reagents are highly toxic and also unstable, since ionic cyanide undergoes base-promoted hydrolysis to form formamide and formate. Consequently, additional cyanide must be introduced in the reagent to compensate for such hydrolyric degradation. Accordingly, there is a need for a method for hemoglobin determinations which has a rapid turnover and does not require the use of cyanide.
The prior art methods described above are characterized by a pH of below 11.3, the use of ionic cyanide, or nonionic surfactants, and completion time to form the chromogen substantially in excess of 30 seconds.
Also, because of environmental reasons, the presence of ionic cyanide in any type of reagent used for analytical purposes is highly undesirable. The disposal of the reaction mixture, or effluent, may require special treatment to reduce the concentration of the ionic cyanide within prescribed limits. Otherwise, if in excess of such limits, disposal of the effluent will require special measures, which are costly. Obviously, effluent disposal is very much simplified if no ionic cyanide, or other toxic material, is present in the effluent.